Mancini G.,Inter University Consortium for the Application of Super Computing for Universities and Research | Mancini G.,Normal School of Pisa | D'Annessa I.,University of Rome Tor Vergata | Coletta A.,University of Rome Tor Vergata | And 4 more authors.PLoS ONE | Year: 2012

Bongiorni S.,University of Tuscia | Mancini G.,Inter University Consortium for the Application of Super Computing for Universities and Research | Chillemi G.,Inter University Consortium for the Application of Super Computing for Universities and Research | Pariset L.,University of Tuscia | Valentini A.,University of TusciaPLoS ONE | Year: 2012

Mancini G.,University of Tuscia | Mancini G.,Inter University Consortium for the Application of Super Computing for Universities and Research | Gargani M.,University of Tuscia | Chillemi G.,Inter University Consortium for the Application of Super Computing for Universities and Research | And 4 more authors.Molecular Biology Reports | Year: 2014

Human topoisomerase I catalyzes the relaxation of DNA supercoils in fundamental cell processes like transcription, replication and chromosomal segregation. It is the only target of the camptothecin family of anticancer drugs. Among these, topotecan has been used to treat lung and ovarian carcinoma for several years. Camptothecins reversibly binds to the covalent intermediate DNA-enzyme, stabilizing the cleavable complex and reducing the religation rate. The stalled complex then collides with the progression of the replication fork, producing lethal double strand DNA breaks and eventually cell death.Long lasting molecular dynamics simulations of the DNA-topoisomerase I binary complex and of the DNA-topoisomerase-topotecan ternary complex have been performed and compared. The conformational space sampled by the binary complex is reduced by the presence of the drug, as observed by principal component and cluster analyses. This conformational restraint is mainly due to the reduced flexibility of residues 633-643 (the region connecting the linker to the core domain) that causes an overall mobility loss in the ternary complex linker domain. During the simulation, DNA/drug stacking interactions are fully maintained, and hydrogen bonds are maintained with the enzyme. Topotecan keeps the catalytic residue Lys532 far from the DNA, making it unable to participate to the religation reaction. Arg364 is observed to interact with both the B and E rings of topotecan with two stable direct hydrogen bonds. An interesting constrain exerted by the protein on the geometrical arrangement of topotecan is also observed.Atomistic-scale understanding of topotecan interactions with the DNA-enzyme complex is fundamental to the explaining of its poisonous effect and of the drug resistance observed in several single residue topoisomerase mutants. We observed significant alterations due to topotecan in both short-range interactions and long-range protein domain communications.

Long-duration comparative molecular dynamics simulations of the DNA-topoisomerase binary and DNA-topoisomerase-indenoisoquinoline ternary complexes have been carried out. The analyses demonstrated the role of the drug in conformationally stabilizing the protein-DNA interaction. In detail, the protein lips, clamping the DNA substrate, interact more tightly in the ternary complex than in the binary one. The drug also reduces the conformational space sampled by the protein linker domain through an increased interaction with the helix bundle proximal to the active site. A similar alteration of linker domain dynamics has been observed in a precedent work for topotecan but the molecular mechanisms were different if compared to those described in this work. Finally, the indenoisoquinoline keeps Lys532 far from the DNA, making it unable to participate in the religation reaction, indicating that both short- and long-range interactions contribute to the drug poisoning effect.